DNA double-strand break damage and repair assessed by pulsed-field gel electrophoresis.

Abstract

This assay quantifies the amount of DNA double-strand break (DSB) damage in attached cell populations embedded in agarose and assayed for migratory DNA using pulsed-field gel electrophoresis (PFGE) with ethidium bromide staining. The assay can measure pre-existing damage, as well as induction of DSB by chemical (e.g., bleomycin), physical (e.g., X-irradiation), or biological (e.g., restriction enzymes) agents. By incubating the cells under physiological conditions prior to processing, the cells are allowed to repair DSB, primarily via the process of nonhomologous end joining. The amount of repair, corresponding to the repair capacity of the treated cells, is then quantified by determining the ratio of the fractions of activity released in these repaired lanes in comparison with the total amount of DNA fragmentation following determination of a optimal exposure for maximum initial fragmentation. Repair kinetics can also be analyzed through a time-course regimen.